Nunc maxisorp 96 wells plates

Gametocyte and asexual extracts were prepared as described previously (21 (link)). Nunc MaxiSorp™ 96-wells plates (ThermoFisher) were coated overnight at 4⁰C with 100 µl, equivalent to 75,000 gametocytes or 40,000 asexual parasites, per well. Plates were blocked with 5% skimmed milk in PBS and subsequently incubated with an 1:50 dilution of citrate plasma. Detection was done with 1:40,000 dilution Goat anti-Human IgG HRP (Invitrogen, Cat. No. 31412). ELISAs were developed by adding 100 µL tetramethylbenzidine and stopped with 50 µL 0.2M H₂SO₄. Absorbances were read at 450nm on an iMark™ microplate absorbance reader (Bio-Rad).
ELISA analyses were performed using Auditable Data Analysis and Management System for ELISA (ADAMSEL FPL v1.1). We included serial diluted control serum from a Dutch missionary that experienced many malaria episodes as a standard curve. The standard curve was plotted on a logarithmic scale and fitted to a power trend line (R2> 0.99), optical density (OD) measurements for each test sample (average of duplicates that were no more than 25% different) were converted to arbitrary units (AU) relative to the control serum, where undiluted control serum was defined to contain 100 AU of IgG.

The domain-specificity of the human monoclonal antibodies was tested in ELISAs with recombinant full-length Pfs48/45 and the three Pfs48/45 fragments. Nunc MaxiSorp™ 96-wells plates (ThermoFisher) were coated with 0.5 μg/ml of recombinant protein overnight at 4°C. Plates were blocked with 5% skimmed milk in PBS before incubation with 10 μg/ml mAb in 1% milk/PBST. Detection was performed by incubation with 1:60,000 Goat anti-Human IgG/HRP-conjugated antibody (Pierce, Cat. No. 31412) in 1% milk/PBST. ELISAs were developed using 100 μl TMB and the reaction was stopped with 50 μL 0.2M H₂SO₄. Absorbance was measured at 450 nm using an iMark™ Microplate Absorbance Reader (Bio-Rad). mAbs were considered positive when the absorbance was higher than the mean absorbance plus three standard deviations of seven negative mAbs.

Nunc MaxiSorp™ 96-wells plates (ThermoFisher) were coated overnight at 4 °C with 100 μl, equivalent to the lysate of 40,000 Pf sporozoites, per well. Plates were blocked with 5% skimmed milk in PBS and subsequently incubated with a 1:100 dilution of sera. Detection was done with 1:40,000 dilution Goat anti-Human IgG HRP (Invitrogen, Cat. No. 31412). ELISAs were developed by adding 100 μL tetramethylbenzidine and stopped with 50 μL 0.2 M H2SO4. Absorbances were read at 450 nm on an iMark™ microplate absorbance reader (Bio-Rad).
ELISA analyses were performed using Auditable Data Analysis and Management System for ELISA (ADAMSEL FPL v1.1).
A pool of 100 sera from adults living in an area in Tanzania where malaria is highly endemic served as positive control serum. The standard curve was plotted on a logarithmic scale and fitted to a power trend line (R2 > 0.99). Optical density (OD) measurements for each test sample were converted to arbitrary units (AU) relative to the control serum (control serum was set at 100) and normalized for the Xmid of each plate.